Toxoplasma gondii is a serious pathogen of humans and livestock in the U.S.A. and world-wide. In addition to its well-known pathogenesis in the developing fetus, in recent years this protozoan parasite has increased its notoriety through the fatal disease it can cause in AIDS patients. Currently, there is no vaccine for Toxoplasma that is designed to impact human health and no drug capable of eliminating the persistent, chronic infection. Disease in AIDS patients is thought to largely result from the reactivation of a chronic infection that persists through the ability of the parasite to differentiate from the actively dividing tachyzoite stage to an encysted bradyzoite stage. The resulting tissue destruction, particularly in the brain and lungs can lead to severe disease or even death. Our goal is to identify and characterize parasite genes (and their respective products) that are critical for the differentiation of tachyzoites to bradyzoites. To identify these genes, we will use a combination of genetic and molecular techniques. For the molecular analysis, we will generate and analyze an expanded set of expressed sequence tags (ESTs) from differentiating and mature bradyzoites. These will be combined with tachyzoite ESTs and used to create microarrays that will allow us to monitor changes in gene expression over a time-course of differentiation. The importance of these genes in differentiation will be assessed by generating and analyzing knock-out mutants for those genes that are of special interest. To identify critical genes whose transcript levels may not change and thus which won't be revealed by the microarrays, we will generate mutants that are not able to differentiate from tachyzoite to bradyzoite. This will be done using direct selection for parasites that fail to express a selectable marker (GFP) under control of a bradyzoite-specific promoter (LDH2) and/or by screening for signature-tagged mutants that fail to complete the asexual cycle in vivo. The protein products of these genes will be characterized with respect to their developmental control and intracellular localization. In this way, we expect to learn what pathways are key to differentiation and growth of bradyzoites. Ultimately, our work will lay the foundation for generation of an animal vaccine that produces a self-limiting infection that cannot be transmitted to other animals or humans. Such a vaccine would break the zoonotic cycle of animal to human transmission.